Article

The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA

London Research Institute, Cancer Research UK, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 10/2010; 17(10):1263-5. DOI: 10.1038/nsmb.1905
Source: PubMed

ABSTRACT Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA2 were observed by EM. BRCA2 directed the binding of RAD51 recombinase to ssDNA, reduced the binding of RAD51 to duplex DNA and stimulated RAD51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA2 in the maintenance of genome stability.

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    • "Our observations, together with previously reported findings, suggest that Rad51 paralogs perform two distinct functions to promote HR: they protect Rad51-ssDNA filaments against disruption by antirecombinases (Liu et al., 2011a) and directly stimulate the intrinsic recombinase activity of Rad51 by remodeling pre-synaptic filaments to an active, ''open,'' flexible, and stable conformation primed for homology search and strand invasion . Importantly, the mechanism we have discovered for the Rad51 paralogs in stimulating HR is distinct from that proposed for other positive regulators of HR, which are epistatic to Rad51 paralogs, including BRCA2 and Rad54 (Chun et al., 2013; Jensen et al., 2010, 2013; Liu et al., 2010; Solinger et al., 2002; Thorslund et al., 2010). We therefore propose a model (Figure 7I) for HR, in photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x 0 is the mean FRET, s is the distribution width, and c 2 quantifies the quality of the fit. "
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    ABSTRACT: Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell 07/2015; 162(2):271-286. DOI:10.1016/j.cell.2015.06.015 · 33.12 Impact Factor
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    • "Our observations, together with previously reported findings, suggest that Rad51 paralogs perform two distinct functions to promote HR: they protect Rad51-ssDNA filaments against disruption by antirecombinases (Liu et al., 2011a) and directly stimulate the intrinsic recombinase activity of Rad51 by remodeling pre-synaptic filaments to an active, ''open,'' flexible, and stable conformation primed for homology search and strand invasion . Importantly, the mechanism we have discovered for the Rad51 paralogs in stimulating HR is distinct from that proposed for other positive regulators of HR, which are epistatic to Rad51 paralogs, including BRCA2 and Rad54 (Chun et al., 2013; Jensen et al., 2010, 2013; Liu et al., 2010; Solinger et al., 2002; Thorslund et al., 2010). We therefore propose a model (Figure 7I) for HR, in photobleaches; histogram of FRET values collected from all molecules with a Gaussian fit are shown in red, where x 0 is the mean FRET, s is the distribution width, and c 2 quantifies the quality of the fit. "
    Cell 07/2015; 162:271-286. · 33.12 Impact Factor
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    • "In the context of HR, the main function of BRCA2 is to mediate this loading [Yuan et al., 1999]. Biochemical assays have shown that the purified full-length BRCA2 protein binds about six molecules of RAD51, targets RAD51 loading onto ssDNA leading to displacement of RPA, and promotes the DNA repair activity of RAD51 [Jensen et al., 2010; Liu et al., 2010; Thorslund et al., 2010]. This region also harbors one NES (residues 1383–1393) necessary for centrosome localization of BRCA2 [Han et al., 2008]. "
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    ABSTRACT: Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore, alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication, and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review, we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including variants of uncertain significance analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk.
    Human Mutation 02/2014; 35(2):151-64. DOI:10.1002/humu.22478 · 5.05 Impact Factor
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